Effects of various chemical modifications of prostacyclin (PGI2) on anti-aggregation on human platelets and on elevation of platelet cyclic AMP (cAMP) were investigated utilizing 2H-adenine labeled human platelet-rich plasma. Elongation of the chain length of PGI2 by one methyl group (20-methyl PGI2) did not markedly alter the PGI2 action on platelets while reducing this chain length by one methyl group (2-nor-methyl PGI2) abolished the PGI2 action on platelets in regards to both anti-aggregatory activity as well as increase in cAMP. Acetylenic analogs of PGI2 were more active than PGI2 and the increase of chain length by one methyl group (20 methyl 13,14 didehydr-PGI2) markedly potentiated the activity against platelet aggregation as well on cAMP while introduction of a methyl group at carbon 16 markedly reduced both activities. Replacement of oxygen in the heterocyclic ring of PGI2 by a methylene group increased the chemical stability but markedly reduced the activity against aggregation as well as cAMP. Similar results were obtained with all analogs of PGI2 when tested on platelet membrane adenylate cyclase.